Recombinant antigen protein, recombinant vector, recombinant host cell, application of recombinant antigen protein, recombinant vector and recombinant host cell, and vaccine

Abstract

The invention discloses a recombinant antigen protein, a recombinant vector, a recombinant host cell and application thereof and a vaccine, and relates to the technical field of antigen preparation, the recombinant antigen protein comprises a capsid protein of porcine circovirus type 2 and a polypeptide in a capsid protein of porcine circovirus type 3; the polypeptide in the porcine circovirus type 3 capsid protein comprises B cell neutralization site polypeptide and / or T cell neutralization site polypeptide. The recombinant antigen protein disclosed by the invention can react with PCV2 type positive serum and PCV3 type positive serum at the same time; the vaccine prepared by mixing the recombinant antigen protein with an adjuvant is an efficient bivalent vaccine, and can effectively stimulate a pig body to generate high-concentration specific antibodies of PCV2 and PCV3, so that a reference is provided for joint prevention of the two viruses. The recombinant antigen protein has the characteristics of stable expression, high yield and good immunogenicity.

Description

Technical Field

[0001] This invention relates to the field of antigen preparation technology, and in particular to a recombinant antigen protein, a recombinant vector, a recombinant host cell, their applications, and vaccines. Background Technology

[0002] Porcine circovirus (PCV) belongs to the genus Porcine circovirus. The virus particle is 17 nm in diameter, has an icosahedral symmetry structure, is non-enveloped, and contains covalently closed single-stranded circular negative-sense DNA. The genome size is approximately 1.7 kb. Four genotypes of PCV have been reported. PCV can co-infect pigs with various viruses and bacteria, exacerbating clinical symptoms. PCV2 and PCV3 (PCV2+3) have been confirmed as major pathogens of porcine circovirus associated disease (PCVAD), which is widespread globally and causes severe economic losses to the pig industry.

[0003] Currently, vaccination is the primary means of preventing PCVAD, and genetically engineered subunit vaccines are highly favored in the market due to their good safety profile. The ORF2 gene of PCV2+3 encodes the icosahedral viral capsid protein (Cap), which is the main antigen of porcine circovirus. However, existing subunit vaccines have insufficient immunogenicity. This may be due to the low full-length expression level of PCV3 Cap and its weak immunogenicity of neutralizing epitope antigens; furthermore, there is currently a lack of measures that can simultaneously prevent infection by PCV2 and PCV3. Summary of the Invention

[0004] The main objective of this invention is to propose a recombinant antigen protein, a recombinant vector, a recombinant host cell, their applications, and a vaccine, aiming to solve the problem of the lack of effective vaccines in the prior art for simultaneously preventing PCV2 and PCV3 infections.

[0005] To achieve the above objectives, the present invention proposes a recombinant antigen protein, which comprises: the capsid protein of porcine circovirus type 2 and a polypeptide in the capsid protein of porcine circovirus type 3. The polypeptides in the porcine circovirus type 3 capsid protein include B cell neutralization site polypeptides and / or T cell neutralization site polypeptides.

[0006] In one embodiment, the porcine circovirus type 2 genotype includes one or more of types 2a, 2b, 2c, 2d, and 2e.

[0007] In one embodiment, the amino acid sequence of the capsid protein of porcine circovirus type 2 is as shown in SEQ ID No. 1; and / or, The amino acid sequence of the B cell neutralization site polypeptide includes at least one of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5; and / or, The amino acid sequence of the T cell neutralization site polypeptide includes at least one of SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

[0008] In one embodiment, the polypeptides in the porcine circovirus type 3 capsid protein include B-cell neutralization site polypeptides and T-cell neutralization site polypeptides: The amino acid sequence of the B cell neutralization site polypeptide includes SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, wherein SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5 are linked by at least one flexible polypeptide; The amino acid sequence of the T cell neutralization site polypeptide includes SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, wherein SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 are linked by at least one flexible polypeptide; The capsid protein of porcine circovirus type 2 and the polypeptide in the capsid protein of porcine circovirus type 3 are linked by at least one flexible polypeptide.

[0009] In one embodiment, the amino acid sequence of the polypeptide in the porcine circovirus type 3 capsid protein is shown in SEQ ID No. 9; and / or, The recombinant antigen protein further includes a TorA signal peptide, the amino acid sequence of which is shown in SEQ ID No. 11; and / or, The amino acid sequence of the recombinant antigen protein is shown in SEQ ID No. 13.

[0010] The present invention also provides a recombinant vector for encoding the aforementioned recombinant antigen protein, the recombinant vector comprising a nucleotide sequence encoding a capsid protein of porcine circovirus type 2 and a nucleotide sequence encoding a polypeptide in the capsid protein of porcine circovirus type 3.

[0011] In one embodiment, the nucleotide sequence encoding the capsid protein of porcine circovirus type 2 is shown in SEQ ID No. 2; and / or, The nucleotide sequence of the polypeptide encoding the capsid protein of porcine circovirus type 3 is shown in SEQ ID No. 10; and / or, The recombinant vector further includes a nucleotide sequence encoding a TorA signal peptide, the nucleotide sequence of which is shown in SEQ ID No. 12; and / or, The nucleotide sequence of the recombinant vector is shown in SEQ ID No. 14.

[0012] The present invention also provides a recombinant host cell, wherein the recombinant host cell comprises the aforementioned recombinant vector.

[0013] The present invention also provides the use of the aforementioned recombinant antigen protein, or the aforementioned recombinant vector, or the aforementioned recombinant host cell in the preparation of a formulation that can induce an immune response in an organism against both porcine circovirus type 2 and porcine circovirus type 3.

[0014] The present invention also provides a vaccine comprising an adjuvant and the aforementioned recombinant antigen protein.

[0015] In this invention, polypeptides from the capsid protein of porcine circovirus type 3 (including B-cell neutralization site polypeptides and / or T-cell neutralization site polypeptides) are assembled with the capsid protein of porcine circovirus type 2 to form a recombinant antigen protein. This recombinant antigen protein can react simultaneously with both PCV2-positive and PCV3-positive sera. The vaccine prepared by mixing this recombinant antigen protein with an adjuvant is a highly effective bivalent vaccine that can effectively stimulate pigs to produce high concentrations of PCV2 and PCV3-specific antibodies, thus providing a reference for the combined prevention of these two viruses. The recombinant antigen protein provided by this invention can self-assemble into virus-like particles, morphologically identical or similar to virus particles. It can be efficiently delivered to the immune system, stimulate efficient uptake by dendritic cells, and strongly stimulate the body's immune system, producing a good immune protective response. This recombinant antigen protein is characterized by stable expression, high yield, and good immunogenicity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 The following are SDS-PAGE images of recombinant antigen protein expression and purification in Example 2 of this invention (lanes: M: Marker; 1: 5-fold dilution of purified protein; 2: original purified protein; 3: purified washed sample; 4: Ni column flow-through; 5: original expression supernatant). Figure 2This is a reaction diagram of recombinant antigen protein and porcine circovirus type 2 positive serum in Example 2 of the present invention (lanes: 1: Marker; 2: Recombinant antigen protein; 3: Negative control). Figure 3 This is a reaction diagram of recombinant antigen protein and porcine circovirus type 3 positive serum in Example 2 of the present invention (lanes: 1: Marker; 2: Recombinant antigen protein; 3: Negative control). Figure 4 This is a transmission electron microscope image of the recombinant antigen protein in Example 2 of the present invention; Figure 5 This is a graph showing the detection level of porcine circovirus type 2 antibody after immunizing pigs with recombinant antigen protein in Example 3 of the present invention; Figure 6 This is a graph showing the detection level of porcine circovirus type 3 antibody after immunizing pigs with recombinant antigen protein in Example 3 of the present invention.

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Porcine circovirus (PCV) belongs to the genus Porcine circovirus. The virus particle is 17 nm in diameter, has an icosahedral symmetry structure, is non-enveloped, and contains covalently closed single-stranded circular negative-sense DNA. The genome size is approximately 1.7 kb. Four genotypes of PCV have been reported. PCV can co-infect pigs with various viruses and bacteria, exacerbating clinical symptoms. PCV2 and PCV3 (PCV2+3) have been confirmed as major pathogens of porcine circovirus associated disease (PCVAD), which is widespread globally and causes severe economic losses to the pig industry.

[0021] Currently, vaccination is the primary means of preventing PCVAD, and genetically engineered subunit vaccines are highly favored in the market due to their good safety profile. The icosahedral viral capsid protein (Cap) encoded by the ORF2 gene of PCV2+3 is the main antigen of porcine circovirus, but existing subunit vaccines have insufficient immunogenicity. Specifically, the PCV3 gene sequence shows low homology with other porcine circoviruses (approximately 40% homology with the PCV2 nucleotide sequence), and existing porcine circovirus vaccines offer no cross-protection against PCV3. There are no reports of successful in vitro culture of PCV3, making it difficult to study its pathogenesis, protein function, and vaccine development. Furthermore, the full-length PCV3 Cap can self-assemble into icosahedral virus-like particles, but the protein expression level of the full-length Cap is low, possibly due to the high arginine content in the nuclear localization signal region of the PCV3 Cap protein. In addition, measures that can simultaneously prevent infection by both PCV2 and PCV3 are currently lacking.

[0022] In view of this, the present invention provides a recombinant antigen protein comprising: a capsid protein of porcine circovirus type 2 and a polypeptide in the capsid protein of porcine circovirus type 3; wherein the polypeptide in the capsid protein of porcine circovirus type 3 includes a neutralization site polypeptide in B cells and / or a neutralization site polypeptide in T cells.

[0023] In the technical solution of this invention, polypeptides from the capsid protein of porcine circovirus type 3, including neutralization site polypeptides in B cells and / or T cell neutralization site polypeptides, are assembled with the capsid protein of porcine circovirus type 2 to form a recombinant antigen protein. This recombinant antigen protein can react simultaneously with PCV2-positive sera and PCV3-positive sera. The vaccine prepared by mixing this recombinant antigen protein with an adjuvant is a highly effective bivalent vaccine that can effectively stimulate pigs to produce high concentrations of PCV2 and PCV3-specific antibodies, thus providing a reference for the combined prevention of the two viruses. The recombinant antigen protein provided by this invention can self-assemble into virus-like particles, which are morphologically identical or similar to virus particles. It can be efficiently delivered to the immune system, stimulate efficient uptake by dendritic cells, and strongly stimulate the body's immune system to produce a good immune protective response. This recombinant antigen protein has the characteristics of stable expression, high yield, and good immunogenicity.

[0024] It is understandable that B cell neutralization sites refer to the short peptide sequences corresponding to antigenic epitopes that can induce or be recognized by B cells and produce neutralizing antibodies. Once recognized by antibodies, these epitopes can block the virus's ability to infect host cells. The definition of T cell neutralization sites is similar.

[0025] It should be noted that this invention displays the neutralizing epitope of PCV3 Cap on the surface of PCV2 Cap particles, meaning that PCV2 Cap is a backbone protein. Furthermore, the DC3 short peptide can be used to promote immune activation and enhance the body's response to antigen stimulation. At the same time, it can also combine with the receptors on the surface of porcine dendritic cells (DCs) to precisely deliver antigens to the MHC-I / MHC-II cross-presentation pathway, thereby enhancing the presentation efficiency of recombinant antigen proteins, improving their immunogenicity, and achieving the goal of simultaneously preventing and controlling PCV2+3.

[0026] In some embodiments, the genotype of porcine circovirus type 2 includes one or more of types 2a, 2b, 2c, 2d, and 2e. Currently, the detection rate of type 2d is higher than that of type 2b in pig farming; therefore, the genotype of porcine circovirus type 2 is preferably type 2d.

[0027] In some embodiments, the amino acid sequence of the capsid protein of porcine circovirus type 2 is shown in SEQ ID No. 1. It should be noted that a dodecimal DC3 sequence is introduced into this amino acid sequence to enhance antigen presentation. Specifically, the DC3 sequence is tandemly linked to the surface loop region (composed of 70 amino acids) of the PCV2-Cap protein via a flexible linker (GGSGG) to facilitate the binding of the DC3 sequence to dendritic cells (DCs).

[0028] In some embodiments, the amino acid sequence of the B cell neutralizing site polypeptide includes at least one of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5. It is understood that the B cell neutralizing site polypeptide can be any one of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, or two or three of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, all within the scope of protection of this invention. Selecting the above-mentioned B cell neutralizing site polypeptide is beneficial for the efficient induction of PCV3 antibody responses by recombinant antigen proteins.

[0029] In some embodiments, the amino acid sequence of the T-cell neutralizing site polypeptide includes at least one of SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8. It is understood that the T-cell neutralizing site polypeptide can be any one of SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, or two or three of SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, all within the scope of protection of this invention. Selecting the above-mentioned T-cell neutralizing site polypeptide is beneficial for the efficient induction of PCV3 antibody responses by recombinant antigen proteins.

[0030] In some embodiments, the polypeptides in the porcine circovirus type 3 capsid protein include a B-cell neutralization site polypeptide and a T-cell neutralization site polypeptide: the amino acid sequence of the B-cell neutralization site polypeptide includes SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, wherein SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5 are linked by at least one flexible polypeptide; the amino acid sequence of the T-cell neutralization site polypeptide includes SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, wherein SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 are linked by at least one flexible polypeptide; and the polypeptides in the porcine circovirus type 2 capsid protein and the porcine circovirus type 3 capsid protein are linked by at least one flexible polypeptide. It is understood that multiple neutralization site peptides in B cells can be linked by flexible peptides, preferably KK; multiple neutralization site peptides in T cells can be linked by flexible peptides, preferably AAY; neutralization site peptides in B cells and neutralization site peptides in T cells can be linked by flexible peptides, preferably GGSGG; preferably, the amino acid sequence of the peptide in the porcine circovirus type 3 capsid protein is shown in SEQ ID No. 9. Furthermore, peptides in the capsid proteins of PCV2 and PCV3 can be linked by flexible peptides, preferably GGSGG. Using flexible peptides for linking these sequences helps ensure the normal expression and function of neutralization sites in B cells, T cells, and the PCV2 capsid protein.

[0031] In some embodiments, the recombinant antigen protein further includes a TorA signal peptide, the amino acid sequence of which is shown in SEQ ID No. 11. Introducing the TorA signal peptide sequence at the N-terminus of the recombinant antigen protein sequence enhances the solubility and native conformation formation of the recombinant protein, thereby increasing the yield, immunoreactivity, and efficacy of the recombinant protein in *E. coli*.

[0032] Most preferably, the amino acid sequence of the recombinant antigen protein is shown in SEQ ID No. 13. This invention utilizes the self-assembly of the PCV2-Cap protein into an icosahedron, linking the three B-cell sites of the PCV3-Cap protein via KK tandem and the three T-cell sites via AAY tandem. Each sequence is then tandemly attached to the icosahedral backbone surface of the PCV2-Cap protein via GGSGG or EAAAk, constructing a virus-like particle antigen and enhancing the immune response to the PCV3 site antigen. Simultaneously, leveraging the ability of DC3 to enhance antigen presentation by DC cells and activate innate immunity, the DC3 sequence is tandemly attached to the surface loop region (70 amino acids) of the PCV2-Cap protein via flexible linker (GGSGG), allowing it to be better displayed on the icosahedral surface, enhancing its binding to DC cells, and improving the immune response to the recombinant antigen.

[0033] The present invention also provides a recombinant vector for encoding the aforementioned recombinant antigen protein, the recombinant vector comprising a nucleotide sequence encoding a capsid protein of porcine circovirus type 2 and a nucleotide sequence encoding a polypeptide in the capsid protein of porcine circovirus type 3. Preferably, the nucleotide sequence encoding the capsid protein of porcine circovirus type 2 is shown in SEQ ID No. 2; and the nucleotide sequence encoding the polypeptide in the capsid protein of porcine circovirus type 3 is shown in SEQ ID No. 10. In some embodiments, the recombinant vector further includes a nucleotide sequence encoding a TorA signal peptide, the nucleotide sequence of which is shown in SEQ ID No. 12.

[0034] Most preferably, the nucleotide sequence of the recombinant vector is shown in SEQ ID No. 14.

[0035] The present invention also provides a recombinant host cell, wherein the recombinant host cell comprises the aforementioned recombinant vector. The cell source of the host cell may be *Escherichia coli* BL21(DE3) competent cells. The recombinant host cell possesses all the beneficial effects of the aforementioned recombinant vector, which will not be elaborated further here.

[0036] This invention also provides the use of the aforementioned recombinant antigen protein, or the aforementioned recombinant vector, or the aforementioned recombinant host cell in the preparation of a formulation capable of inducing an immune response in an organism against both porcine circovirus type 2 and porcine circovirus type 3. Therefore, it possesses all the beneficial effects of the aforementioned recombinant antigen protein, or the aforementioned recombinant vector, or the aforementioned recombinant host cell, which will not be elaborated further here. It is understood that the formulation includes vaccines (which may be subunit vaccines), immune enhancers, diagnostic reagents, or immunotherapeutic drugs.

[0037] The present invention also provides a vaccine comprising an adjuvant and the aforementioned recombinant antigen protein. Therefore, it possesses all the beneficial effects of the aforementioned recombinant viral antigen or the aforementioned virus-like particles, which will not be elaborated further here.

[0038] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0039] Example 1: Determination and optimization of the gene sequence of the recombinant antigen protein PCV2-PCV3-Cap 1. Confirmation and optimization of the recombinant gene sequence of PCV2-Cap protein The amino acid sequence of the Cap protein of PCV2 genotype 2d was obtained from GenBank in the National Center for Biotechnology Information (NCBI).

[0040] Sequence optimization steps: (1) The amino acid sequence of the Cap protein of PCV2 type 2d genotype was optimized as follows: The first 40 amino acids at the N-terminus and the 4 amino acids at the C-terminus were deleted. A DC3 sequence was introduced into the deleted amino acid sequence, and a flexible peptide was used to tandemly attach the DC3 sequence to the surface loop region of the PCV2-Cap protein. The optimized amino acid sequence is shown in SEQ ID No. 1. NGIFNTRLSRTIGYTVKKTTVRTPSWNVDGGSGGCRKARSAKHKTAGGSGGMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNF VTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQDYNIRITMYVQFREFNLKDP Specifically, deleting the first 40 amino acids at the N-terminus removes the arginine-rich nuclear localization signal, preventing the expressionable protein from being retained in the nucleus and affecting protein expression in the cytoplasm. It also removes antibody-dependent enhancement (ADE)-related epitopes, reducing the likelihood of ADE. Furthermore, the deleted amino acids have little impact on virus-like particle assembly. DC2 is a 12-mer DC (dendritic cell) targeting peptide, which is a peptide composed of 12 amino acids. Its introduction can increase antigen presentation. The surface loop region of the PCV2-Cap protein consists of 70 amino acids. Connecting DC3 to its N-terminus is beneficial for the structural stability of the recombinant antigen protein and the binding of DC3 to DC cells.

[0041] (2) Based on the amino acid sequence of SEQ ID No. 1 and the preferences of the E. coli expression system, the encoding nucleotide sequence was designed and optimized, and the AU-enriched region in the nucleotide sequence was optimized to obtain SEQ ID No. 2: AACGGCATCTTTAACACCCGCCTGAGCCGCACCATTGGCTATACCGTGAAAAAAACCACCGTGCGCACCCCGAGCTGGAACGTGGATGGCGGCAGCGGCGGCTGCCGCAAAGCGCGCAGCGCGAAACATAAAACCGCGGGCGGCAGCGGCGGCATGATGCGCTTT AAATTAACGACTTTCTGCCGCCGGGCGGCGGCAGCAACCCGCTGACCGTGCCGTTTGAATATTATCGCATTCGCAAAGTGAAAGTGGAATTCTGGCCGTGCAGCCCGATTACCCAGGGCGATCGCGGCGTGGGCAGCACCGCGGTGATTCTGGATGATAACTTTG TGACCAAAGCGAACGCGCTGACCTATGATCCGTATGTGAACTATAGCAGCCGCCATACCATTACCCAGCCGTTTAGCTATCATAGCCGCTATTTCACCCCGAAACCGGTGCTGGATCGCACCATTGATTACTTTCAGCCGAACAACAAACGCAACCAGCTGTGGCT GCGCCTGCAGACCACCGGCAACGTGGATCATGTGGGCCTGGGCACCGCGTTTGAAAACAGCATTTATGATCAGGATTATAACATTCGCATTACCATGTATGTGCAGGATTATAACATTCGCATTACCATGTATGTGCAGTTTCGCGAATTTAACCTGAAAGATCCG 2. Confirmation and optimization of the recombinant gene sequence of PCV3-Cap protein Based on the amino acid sequence of PCV3-type Cap protein in GenBank, the following operations were performed using tools such as SignalP and ABCpred: (1) Determine the sequence of PCV3-Cap 73 ETAISFEYYKILKMK 87 (SEQ ID No. 3) 161 QSLFFFSRPTPWLNT 175 (SEQ ID No. 4) and 201 YGTKEVWIRYKSVL 214 (SEQ ID No. 5) These three B cell neutral epitopes are tandemly linked together using KK; (2) Determine the sequence of PCV3-Cap 154 TTSAHPGQSLFFFS 167 (SEQ ID No. 6) 161 QSLFFFSRPTPWLNT 175 (SEQ ID No. 7) and 185 LLWSIYVPEKTGMTD 199 (SEQ ID No. 8) These three T cell neutral epitopes are linked together with AAY; (3) The B cell neutralization epitopes and T cell neutralization epitopes obtained above were concatenated via GGSGG to obtain SEQ ID No. 9: ETAISFEYYKILKMKKKQSLFFFSRPTPWLNTKKYGTKEVWIRYKSVLGGSGGTTSAHPGQSLFFFSAAYQSLFFFSRPTPWLNTAAYLLWSIYVPEKTGMTD Based on the amino acid sequence of SEQ ID No. 9 and the preferences of the E. coli expression system, the encoding nucleotide sequence was designed and optimized, and the AT-enriched region in the nucleotide sequence was further optimized to obtain SEQ ID No. 10: GAAACCGCGATTAGCTTTGAATATTATAAAATTCTGAAAATGAAAAAAAAACAGAGCCTGTTTTTTTTTAGCCGCCCGAACCCCGTGGCTGAACACCAAAAAATATGGCACCAAAGAAGTGTGGATTCGCTATAAAAGCGTGCTGGGCGGCAGCG GCGGCACCACCAGCGCGCATCCGGGCCAGAGCCTGTTTTTTTTTAGCGCGGCGTATCAGAGCCTGTTTTTTTTTAGCCGCCCGACCCCGTGGCTGAACACCGCGGCGTATCTGCTGTGGAGCATTTATGTGCCGGAAAAAAACCGGCATGACCGAT 3. Determination and optimization of the TorA signal peptide gene sequence By utilizing the TorA signal peptide's beneficial properties for protein structure formation and transport, the TorA signal peptide sequence is introduced at the N-terminus of the recombinant antigen protein sequence to enhance the solubility and native conformation formation of the recombinant antigen protein, thereby increasing the yield, immunoreactivity, and effectiveness of the recombinant antigen protein in Escherichia coli.

[0042] The amino acid sequence of the introduced TorA signal peptide is shown in SEQ ID No. 11: MNNNDLFQASRRRFLAQLGLGLTVAGMLAQA Based on the amino acid sequence of SEQ ID No. 11 and the preferences of the E. coli expression system, the encoding nucleotide sequence was designed and optimized. To facilitate its expression, the optimized nucleotide sequence is shown in SEQ ID No. 12: ATGAACAACAACGATCTGTTTCAGGCGAGCCGCCGCCGCTTTCTGGCGCAGCTGGGCGGCCTGACCGTGGCGGGCATGCTGGCCAGGCG 4. Determination and optimization of the gene sequence of recombinant antigen protein Introducing a 6×His tag at the C-terminus of the recombinant antigen protein facilitates purification. The PCV2-Cap protein backbone is tandemly linked to the TorA signal peptide via EAAAK, and to the B and T cell neutralization sites of PCV3-Cap, the DC3 short peptide, and His via flexible GGSGG links. Therefore, the target linking sequence of the recombinant antigen protein is as follows: TorA signal peptide sequence - first part of the PCV2-Cap recombinant sequence - DC3 short peptide - second part of the PCV2-Cap recombinant sequence - PCV3-Cap recombinant sequence - His tag sequence. The first part of the PCV2-Cap recombinant sequence - DC3 short peptide - second part of the PCV2-Cap recombinant sequence constitutes the aforementioned PCV2-Cap recombinant sequence. The determined amino acid sequence of the recombinant antigen protein is shown in SEQ ID No. 13. MNNNDLFQASRRRFLAQLGGLTVAGMLAQAEAAAKNGIFNTRLSRTIGYTVKKTTVRTPSWNVDGGSGGCRKARSAKHKTAGGSGGMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTI DYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQDYNIRITMYVQFREFNLKDPGGSGGETAISFEYYKILKMKKKQSLFFFSRPTPWLNTKKYGTKEVWIRYKSVLGGSGGTTSAHPGQSLFFFSAAYQSLFFFSRPTPWLNTAAYLLWSIYVPEKTGMTDGGSGGHHHHHH Based on the amino acid sequence of SEQ ID No. 13 and the preferences of the E. coli expression system, the encoding nucleotide sequence was designed and optimized. To facilitate expression, NcoI and XhoI restriction sites were introduced before and after the sequence, respectively, and a stop codon was introduced between the C-terminal restriction sites. The AU-enriched region of the full nucleotide sequence was optimized. The final nucleotide sequence of the recombinant antigen protein PCV2-PCV3-Cap is shown in SEQ ID No. 14. Example 2: Preparation of virus-like particles from recombinant antigen protein PCV2-PCV3-Cap 1. Preparation of recombinant vectors Following the nucleotide sequence encoding the recombinant antigen protein obtained in Example 1, the nucleotide sequence PCV2-PCV3-Cap (SEQ ID No. 8) was artificially synthesized and used in pET. Using the 28a (+) plasmid as a vector, the nucleotide sequence encoding the recombinant antigen protein PCV2-PCV3-Cap was cloned and recombined into pET. In plasmid 28a (+), the recombinant plasmid pET-PCV2-PCV3-Cap was constructed.

[0043] 2. Transformation and induction of protein expression The recombinant plasmid pET-PCV2-PCV3-Cap obtained in step 1 was transformed into *E. coli* BL21(DE3) competent cells. The main procedures are as follows: After thawing Escherichia coli BL21(DE3) competent cells on ice, add 5 μL of the recombinant plasmid obtained in step 1 (2 ng / mL), mix well, and place on ice for 30 min; heat shock in a 42℃ water bath for 30 s, place on ice for 5 min, add 500 μL of culture medium, mix well, and incubate at 37℃ and 200 rpm for 1 h to obtain the cultured bacterial solution; Take 100 μL of the cultured bacterial solution and spread it evenly on an LB agar plate containing 50 μg / mL kanamycin. Incubate at 37°C with the plate upside down overnight to obtain the cultured colonies. Single colonies were selected for colony PCR and sequencing identification to ensure correct transformation and obtain correctly transformed strains. Select the strains that have undergone the correct transformation, inoculate them into LB medium containing 50 μg / mL kanamycin, and culture until approximately OD200. 600 When the concentration of the protein was 0.6~0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the mixture was cultured at 20°C for 12 h to induce the expression of the recombinant antigen protein. Bacterial cultures were collected before and after induction of recombinant antigen protein expression. After ultrasonic disruption, samples (supernatant) were taken for SDS-PAGE analysis to detect the expression of the recombinant antigen protein. The SDS-PAGE images of the supernatant from the recombinant antigen protein expression are shown below. Figure 1 Lane 5 in the swimming pool.

[0044] 3. Purification of recombinant antigen proteins and detection of virus-like particles After the bacterial culture in step 2 is induced and expressed, it is broken down and purified to obtain the recombinant antigen protein. The main steps are as follows: Centrifuge the bacterial culture induced in step 2 at 12000 rpm for 15 min to collect the bacterial cells, resuspend the bacterial cells in superphosphate buffer (PBS), and lyse the bacterial cells by sonication on ice. Then centrifuge the culture at 4°C, 12000 rpm for 30 min to collect the supernatant. The supernatant was passed through a nickel (Ni) column and washed with 10 column volumes of 50 mM imidazole followed by elution with 500 mM imidazole to obtain the purified recombinant antigen protein. Western blotting was performed on the purified recombinant antigen protein using positive sera for PCV2 and PCV3 as primary antibodies. The results showed that the recombinant antigen protein reacted with both positive sera. Figure 2 and Figure 3 ).

[0045] The morphology of the assembled nanoparticle vaccine was observed by transmission electron microscopy (TEM) using a negative staining method. Figure 4 It can be seen that recombinant antigen proteins can self-assemble into virus-like particles.

[0046] Example 3: Preparation and application of immunogens based on recombinant antigen proteins Vaccine preparation: The purified PCV2-PCV3-Cap recombinant antigen protein was mixed with Gel02 adjuvant from Seppic Company of France to make the final concentration of the recombinant antigen protein 50 μg / mL. The mixture was stirred for 30 minutes to obtain a bivalent vaccine of porcine circovirus type 2 + type 3 virus-like particles.

[0047] Ten healthy 4-5 week old piglets, both negative for PCV2 antigen and antibody and PCV3 antigen and antibody, were randomly divided into two groups of five each. The first group was the immunization group, with each piglet receiving an intramuscular injection of 2 mL of the prepared bivalent vaccine in the neck. The second group was the control group, receiving no injection. Fourteen days later, the immunized piglets underwent a second immunization in the same manner. Following the first immunization, blood samples were collected weekly from each group to separate serum. Antibody detection kits (Porcine Circovirus Type II Antibody Detection Kit purchased from Shanghai Lianmai Biotechnology Co., Ltd., catalog number: LM-0085LC; Biostone Porcine Circovirus Type 3 Antibody Detection Kit purchased from Shanghai Lianmai Biotechnology Co., Ltd., catalog number: 10086-02 / 05) were used to detect the antibody levels of porcine circovirus type 2 and porcine circovirus type 3 in the serum. Results are as follows: Figure 5 and Figure 6 As shown.

[0048] Figure 5 and Figure 6Antibody detection results showed that the prepared recombinant antigen protein immunogen rapidly produced antibodies in piglets after immunization. The levels of antibodies against porcine circovirus type 2 and type 3 (PCV2) both increased significantly after immunization. Specifically, for PCV2 antibodies: some immunized pigs showed seroconversion 7 days after the first immunization, 4 / 5 of the immunized pigs showed seroconversion 14 days after the first immunization, and all immunized pigs showed seroconversion 21 days after the first immunization, which persisted for up to 6 months post-immunization. For PCV3 antibodies: some immunized pigs showed seroconversion 7 days after the first immunization, 4 / 5 of the immunized pigs showed seroconversion 14 days after the first immunization, and all immunized pigs showed seroconversion 21 days after the first immunization, which persisted for up to 6 months post-immunization. In contrast, the control group piglets showed negative results for both PCV2 and PCV3 antibodies. These results indicate that the PCV2+3 recombinant antigen protein vaccine prepared using the genetically engineered strain of this invention can effectively stimulate the production of PCV2 and PCV3 specific antibodies in pigs, and the antibodies produced after immunization have a long duration of action.

[0049] This application uses PCV2 type Cap as a virus-like particle vector, tandemly incorporating neutralizing epitopes in B cells and T cells of PCV3 type Cap, and utilizes DC3's targeting of DCs to enhance antigen uptake and presentation, effectively stimulating the immune response. A bivalent vaccine of porcine circovirus type 2 and 3 virus-like particle antigens was prepared. Antibody results in piglets with both antigen and antibody negative after mixing with adjuvant showed that this bivalent vaccine can simultaneously induce specific antibodies against PCV2 and PCV3, providing a reference for the combined prevention and treatment of PCV2 and PCV3.

[0050] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A recombinant antigen protein, characterized in that, The recombinant antigen protein includes: the capsid protein of porcine circovirus type 2 and the polypeptide in the capsid protein of porcine circovirus type 3; The polypeptides in the porcine circovirus type 3 capsid protein include B cell neutralization site polypeptides and / or T cell neutralization site polypeptides.

2. The recombinant antigen protein as described in claim 1, characterized in that, The porcine circovirus type 2 genotype includes one or more of the following: type 2a, type 2b, type 2c, type 2d, and type 2e.

3. The recombinant antigen protein as described in claim 1, characterized in that, The amino acid sequence of the capsid protein of porcine circovirus type 2 is shown in SEQ ID No. 1; and / or, The amino acid sequence of the B cell neutralization site polypeptide includes at least one of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5; and / or, The amino acid sequence of the T cell neutralization site polypeptide includes at least one of SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No.

8.

4. The recombinant antigen protein as described in claim 1, characterized in that, The polypeptides in the porcine circovirus type 3 capsid protein include B cell neutralization site polypeptides and T cell neutralization site polypeptides: The amino acid sequence of the B cell neutralization site polypeptide includes SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, wherein SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5 are linked by at least one flexible polypeptide; The amino acid sequence of the T cell neutralization site polypeptide includes SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, wherein SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8 are linked by at least one flexible polypeptide; The capsid protein of porcine circovirus type 2 and the polypeptide in the capsid protein of porcine circovirus type 3 are linked by at least one flexible polypeptide.

5. The recombinant antigen protein as described in claim 4, characterized in that, The amino acid sequence of the polypeptide in the porcine circovirus type 3 capsid protein is shown in SEQ ID No. 9; and / or, The recombinant antigen protein further includes a TorA signal peptide, the amino acid sequence of which is shown in SEQ ID No. 11; and / or, The amino acid sequence of the recombinant antigen protein is shown in SEQ ID No.

13.

6. A recombinant vector, characterized in that, The recombinant vector is used to encode the recombinant antigen protein as described in any one of claims 1 to 5, wherein the recombinant vector comprises a nucleotide sequence encoding a capsid protein of porcine circovirus type 2 and a nucleotide sequence encoding a polypeptide in the capsid protein of porcine circovirus type 3.

7. The recombinant vector as described in claim 6, characterized in that, The nucleotide sequence encoding the capsid protein of porcine circovirus type 2 is shown in SEQ ID No. 2; and / or, The nucleotide sequence of the polypeptide encoding the capsid protein of porcine circovirus type 3 is shown in SEQ ID No. 10; and / or, The recombinant vector further includes a nucleotide sequence encoding a TorA signal peptide, the nucleotide sequence of which is shown in SEQ ID No. 12; and / or, The nucleotide sequence of the recombinant vector is shown in SEQ ID No.

14.

8. A recombinant host cell, characterized in that, The recombinant host cell includes the recombinant vector as described in claim 6 or 7.

9. The use of a recombinant antigen protein as described in any one of claims 1 to 5, or a recombinant vector as described in any one of claims 6 to 7, or a recombinant host cell as described in claim 8, in the preparation of an agent capable of evoking an immune response in an organism against both porcine circovirus type 2 and porcine circovirus type 3.

10. A vaccine, characterized in that, The vaccine includes an adjuvant and a recombinant antigen protein as described in any one of claims 1 to 5.

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